Automated detection of malaria-associated intraleucocytic haemozoin by Cell-Dyn CD4000 depolarization analysis.

Laboratory tests for malaria are only performed if there is clinical suspicion of the disease, and a missed diagnosis contributes substantially to morbidity and mortality. Malaria parasites produce haemozoin, which is able to depolarize light and this allows the automated detection of malaria during routine complete blood count analysis (CBC) with some Abbott Cell-Dyn instruments. In this study, we evaluated the Cell-Dyn CD4000 with 831 blood samples submitted for malaria investigations. Samples were categorized as malaria negative (n = 417), convalescent malaria (n = 64) or malaria positive (n = 350) by reference to thin/thick film microscopy, 'rapid test' procedures, polymerase chain reaction analysis and clinical history. With regard to CD4000 depolarization analysis, a malaria positive CD4000 pattern was ascribed to samples that showed one or more abnormal depolarizing purple events, which corresponded to monocytes containing ingested malaria pigment (haemozoin). Positive CD4000 patterns were observed in 11 of 417, 50 of 64 and 281 of 350 of malaria negative, convalescent malaria and malaria positive samples respectively. The specificity and positive predictive values for malaria (active and convalescent) were very high (97.4 and 96.8%, respectively), while sensitivity and negative predictive values were 80.0 and 83.0% respectively. Depolarization analysis was particularly effective for Plasmodium falciparum malaria but there was lower detection sensitivity for White compared with Black African patients. CD4000 90 degrees depolarization vs 0 degrees analysis revealed a proportion of samples with small nonleucocyte-associated depolarizing particles. Appearance of such events in the form of a discrete cluster was associated with P. vivax rather than P. falciparum infection.

Systematic review of combination antiretroviral therapy with didanosine plus hydroxyurea: a partial solution to Africa's HIV/AIDS problem?

Effective antiretroviral therapy remains beyond the reach of most human immunodeficiency virus (HIV)-infected persons living in the third world because of its tremendous cost. The cancer drug, hydroxyurea, inhibits HIV-1 replication in vitro and, when combined with didanosine (ddI), results in significant antiretroviral synergy. In vivo, hydroxyurea specifically targets quiescent lymphocytes and macrophages, important cellular reservoirs for HIV-1, and the combination of ddI plus hydroxyurea effectively reduces plasma HIV-1 RNA levels. Combination ddI-hydroxyurea costs about one-eighth as much as currently recommended triple drug combinations, and several countries in Africa are exploring the feasibility of widescale use of ddI-hydroxyurea for their HIV-infected populations. Intrigued by its potential relevance for Africa, the authors reviewed the literature on the in vitro and clinical efficacy of ddI plus hydroxyurea against HIV. The combination of ddI plus hydroxyurea is an effective and potentially more affordable regimen for HIV-infected persons living in poorer countries.

Automated malaria detection by depolarization of laser light.

Anecdotal experience with full blood count (FBC) technology incorporating analysis of depolarized laser light (DLL) for the enumeration of eosinophils showed that malaria infection generated unusual distributions in the white cell channels. The objective of this study was to identify and define criteria for a diagnosis of malaria using this technology. To determine sensitivity, specificity, and positive and negative predictive values, 224 directed samples referred specifically for malaria were used; true positives were defined as those in which malaria was identified by microscopic and/or immunological methods. For the DLL method, positive was defined as one or more large mononuclear cell(s) for which the 90 degrees depolarized signal exceeded the 90 degrees polarized signal. To determine possible utility in a routine haematology laboratory setting, 220 random undirected FBC samples were evaluated for possible malaria infection by the DLL method. Of the 224 directed samples, 95 were malaria positive as determined by microscopic and/or immunological methods, and 129 were negative. For the DLL method, overall sensitivity was 72% (90% in the case of Black Africans), and specificity 96%. Positive and negative predictive values overall were 93% and 82% respectively. In the utility study a single positive result was identified among the 220 samples studied. This was found to be from a patient with malaria. The detection of unexpected malaria by automated screening FBC analysis could substantially lower the mortality and morbidity from unascertained infection, especially in indigenous African peoples.

CD4+ T cell enumeration in HIV infection with limited resources.

The incidence of human immunodeficiency virus (HIV) infection continues to increase in South Africa. Limited resources are available for diagnosis and management of the disease and the development of affordable strategies is required. Absolute CD4 counts are used locally predominantly to monitor disease progression and institute prophylaxis against opportunistic infections. A dramatic increase in demand for CD4 counts prompted an investigation for a more cost-effective flow cytometry method than those currently recommended by the Centers for Disease Control (CDC). CD4 counts generated by two different single tube methods using CD3/CD4/CD8 [1(3)] and CD4 [1(1)] antibodies, respectively, were compared to the CDC recommended 6 tube 2 colour panel [6(2)]. Whole blood analysis using the Coulter Multi-Q-Prep system and an Epics XL Flow Cytometer (Coulter, Hialeah, FL) was performed for each of the three methods. Random samples from HIV positive adult patients were compared. A mean difference in the absolute CD4 counts of less than 10x10(6)/l was generated by both of the alternative panels when compared with the 6(2) panel. The precision of the three methods is comparable. In reagents alone, the 1(3) and 1(1) methods represent a cost saving of 76% and 93%, respectively, over the 6(2) method. The 1(3) and 1(1) panels would permit more affordable CD4 counts to be determined by the gold standard methodology of flow cytometry with no clinically significant sacrifices in accuracy or precision.

Identification of endonuclease activity in HIV-1 gp120 preparations produced using baculovirus expression systems.

Experiments undertaken with commercially available recombinantly produced human immunodeficiency virus Type 1 (HIV-1) gp120 demonstrated that the resuspended lyophilized protein, a product of the baculovirus expression system, had intrinsic nuclease activity. This nuclease activity was distinguishable from the molecular-grade bovine serum albumin that it was constituted in. The activity was thermolabile in that if the preparation was heated to 100 degrees C for 10 min, the activity was abolished, although this did not happen when it was stored at -20 degrees C. The nuclease activity was also Ca+2- and Mg+2-dependent, and had endonuclease as opposed to exonuclease activity. Zn+2 ions were found to inhibit the enzyme. The intensity of nuclease activity varied from batch to batch. A lyophilized homogenate of Sf9 insect cells expressing the Rho baculovirus-derived red blood cell protein 4.2 (Pallidin) was also found to have nuclease activity on reconstitution. In contrast, most, though not all E. coli-produced recombinant proteins were found to be free of nuclease activity. The use of activity gels to identify the size of the nuclease contained in the gp120 preparation was limited, because despite the use of many renaturation methods, the enzyme in the gp120 preparation could not be functionally resuscitated following sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunoprecipitation studies were useful to demonstrate that nuclease activity in the gp120 preparation was functionally distinguishable from the gp120 itself. When mononuclear cells transformed with anti-CD3 were concurrently incubated with gp120 (5-40 micrograms/mL), internucleosomal DNA fragments characteristic of apoptosis were demonstrated in the supernatant by DNA gel electrophoresis. In the context of HIV-1 and AIDS, where the depletion of CD4+ T-cells has been found to be associated with apoptosis, nuclease activity intrinsic to the gp120 preparation used in experimentation may potentially alter experimental results.

Evaluation of DNA analysis for evidence of apoptosis in megaloblastic anaemia.

This study involved DNA analysis of bone marrow cells of 15 patients with megaloblastic anaemia. The diagnosis was based on the morphological changes seen in the bone marrow, associated with either a low red cell folate or serum vitamin B12 level and an adequate response to appropriate therapy as confirmation of the diagnosis. Flow cytometric DNA analysis showed an increase in the S and G2 phases of the cell cycle, but conventional agarose gel electrophoretic DNA analysis did not confirm the characteristic 'ladder pattern' which might have been expected in classic apoptosis. In addition, cells showing morphological changes suggestive of apoptosis, such as nuclear condensation and fragmentation, did not show evidence of DNA fragmentation using the ApopTag in situ digoxigenin nucleotide labelled, peroxidase detection system. Further studies using annexin V flow cytometric analysis and pulsed field gel electrophoresis were also unable to detect evidence of apoptosis as a significant cause of cell death in megaloblastic anaemia.

Field evaluation of a novel haemoglobin measuring device designed for use in a rural setting.

OBJECTIVE: To evaluate the use of a robust, cheap method for haemoglobin estimation by non-laboratory-trained personnel in a rural setting. DESIGN: Comparative study. SETTING: Tintswalo Hospital, Acornhoek. PARTICIPANTS: 7 nursing sisters, 4 medical students, 2 lay persons. OUTCOME MEASURES: Haemoglobin estimates obtained with the colour scale were compared with the 'true Hb' values determined by the H x 3 Bayer-Technicon automated blood analyser. RESULTS: Although individuals varied in their abilities to use the colour scale, its performance was generally very good when measured against automated haemoglobinometry, as determined by bias and regression analysis and also in terms of its capacity to detect anaemia, as measured by sensitivity, specificity and positive and negative predictive values. CONCLUSIONS: Haemoglobin estimates obtained with the World Health Organisation colour scale are generally reliable, although cognisance should be taken of individual variability. While the utility of the device in monitoring response to therapy remains to be seen, it promises to be a suitable method for mass screening for anaemia.

Protein synthesis inhibition is not a requisite for puromycin- and cycloheximide-induced c-myc mRNA superinduction.

The phenomenon of superinduction refers to the process by which high doses (30-180 microM) of the protein synthesis inhibitors Puromycin (PM) or Cycloheximide (CHX) augment and stabilize mRNA transcript levels through mechanisms which are dependent on the complete inhibition of protein synthesis. The current study undertaken in HL-60 leukaemic cells has dissociated the protein synthesis-inhibitory effects of PM and CHX from their c-myc mRNA superinducing effects. When employed at concentrations which were sub-inhibitory with respect to protein synthesis (0.2 microM), PM and CHX nevertheless elicited the phenomenon of superinduction, possibly through mechanisms related to intracellular signalling.

Glycophorin variants and Plasmodium falciparum: protective effect of the Dantu phenotype in vitro.

Experimental work was carried out to establish the growth characteristics of Plasmodium falciparum in an in vitro culture system using cells with the Dantu, Henshaw and S-s-U- blood-group variants. A flow cytometric technique, using the dye thiazole orange, was adapted for use on the Epics Profile II flow cytometer to count the parasites. This was performed at 24, 48 and 72 h. The ability of the parasites to grow in red cells of the Dantu and Henshaw phenotypes was also assayed by 3[H] hypoxanthine incorporation. Relative to control red cells, S-s-U- cells and Dantu cells were less suitable as host cells for P. falciparum in vitro. In contrast, cells expressing the Henshaw antigen were equally sensitive to P. falciparum infection as were normal controls. These data support the notion that glycophorins play an important role in P. falciparum infection. Further studies are required to evaluate the epidemiological significance of these results.

Puromycin-elicited c-myc mRNA superinduction precedes apoptosis in HL-60 leukaemic cells.

The phenomenon of superinduction refers to the process by which high concentrations of protein synthesis inhibitors augment and stabilize mRNA transcript levels, e.g. 36-180 microM of Puromycin (PM) has been found to elicit c-myc mRNA superinduction. The expression of the proto-oncogene c-myc has been strongly variously implicated in the regulation of the apoptotic cascade. Since we recently found that a low dose of the protein synthesis inhibitor PM activated the apoptotic cascade in HL-60 leukaemic cells, the current study was undertaken to examine c-myc mRNA transcript levels in such cells, and to assess the relationship, if any, between PM-elicited c-myc mRNA superinduction and subsequent activation of the apoptotic cascade. PM was employed in vitro at doses of 0.9 microM and 2 microM. Dose-dependent c-myc mRNA superinduction was present at 1 hour of PM-exposure, and was associated with subsequent activation of the apoptotic cascade at 24 hours of exposure. Apoptosis was confirmed morphologically as evidenced by chromatin condensation, nuclear fragmentation and the formation of apoptotic bodies, and by DNA agarose gel electrophoresis which showed the pattern of double-stranded DNA fragments that result from the activation of an endogenous endonuclease. [C14]Leucine incorporation studies at 1 hour demonstrated minimal protein synthesis inhibition at doses used, suggesting that c-myc mRNA superinduction in this context may have been the result of mechanisms which were independent of the inhibition of synthesis of new proteins, such as interruptions in signal transduction.

Cell-cycle disruptions and apoptosis induced by the cyclophosphamide derivative mafosfamide.

The effect of the cyclophosphamide derivative mafosfamide (ASTA Z 7557) was investigated in vitro in HL60 leukemic cells. Mafosfamide, which rapidly generates 4-hydroxycyclophosphamide after aqueous dissolution, was employed at doses ranging from 0.1 to 10 micrograms/mL. In unsynchronized cells, mafosfamide exposure was associated with an S-phase accumulation, a suggestion of a G2-phase arrest and morphological and biochemical evidence of apoptosis. In cells that had been synchronized by the double thymidine block method, S-phase progression was considerably delayed in the presence of mafosfamide. The apoptosis that was evident in mafosfamide-treated cells 12 hours after release from the thymidine block was found to occur in the presence of S-phase and G2-phase cell-cycle arrests. Taken together, the current data suggest that mafosfamide may have potential synergism with other anticancer agents that elicit similar cell-cycle arrests as well as with chemotherapeutic drugs that activate the apoptotic cascade.

C-myc expression is down-regulated in mafosfamide-treated HL-60 cells undergoing apoptosis.

The expression of the proto-oncogene c-myc has been strongly implicated in the regulation of apoptosis. Since we have previously shown apoptosis to occur in HL-60 leukemic cells exposed to the prodrug Mafosfamide (ASTA Z 7557), the present study was undertaken to examine the levels of c-myc mRNA transcripts in such cells. Mafosfamide, which is a cyclophosphamide derivative that rapidly generates 4-Hydroperoxycyclophosphamide after aqueous dissolution, was employed in doses ranging from 0.1 to 20 micrograms/ml. No changes in c-myc mRNA levels were evident after 1 hour of exposure to Mafosfamide. After 6 and 24 hours of Mafosfamide-exposure, however, there was a dose- and time-dependent decrease in c-myc transcripts. c-myc mRNA expression was reduced to a greater extent than was either beta-actin of GAPDH expression. Morphological, biochemical and ultrastructural evidence of apoptosis accompanied the Mafosfamide-induced c-myc mRNA down-regulation at 24 hours. We conclude that, in the context of Mafosfamide-treated HL-60 cells, upregulation of c-myc mRNA transcription was not fundamental for the activation of the apoptotic cascade.

Substituted purines elicit differential cytokinetic, molecular and phenotypic effects in HL-60 cells.

This study investigated and compared the cytokinetic, phenotypic and molecular effects elicited in HL-60 human leukemic cells by low doses (0.6 microM) of 3 closely related, substituted purines, puromycin (PM), puromycin aminonucleoside (PAN) and 6-dimethylaminopurine (6-DMAP). PM, but not 6-DMAP or PAN, inhibited [14C]leucine incorporation, induced a time-related cytotoxic effect, a G2-arrest, a metaphase-mitotic-arrest, apoptosis and c-myc mRNA superinduction. PAN and 6-DMAP exerted no detectable morphological or cytokinetic effects, although exposure to these drugs resulted in downregulation of c-myc mRNA levels. We suggest that the specific effects exerted by PM relate to the generation of nascent peptidyl-PM complexes by PM, but not by 6-DMAP or PAN.

CD10 antigen density in childhood common acute lymphoblastic leukaemia: comparisons of race and sex.

During the 25 month period from July 1989 until August 1991, 58 children with FAB defined acute lymphoblastic leukaemia (ALL) were referred for immunophenotypic analysis. Of these, 42 children with a common/pre-B phenotype (CD19/CD10-positive) were studied specifically to assess CD10 antigen density. A pattern of segregation was found between males and females and between black and white children. Black males, who are the worst prognostic group, had the lowest CD10 density, while white females, known to constitute the best prognostic group, had significantly higher CD10 antigen density than the other groups. Black females and white males occupied intermediate positions with respect to CD10 antigen density. A two way analysis of variance showed that although sex had contributed significantly to this variation (p = 0.0038), the contribution of race was marginal (p = 0.0530). It is hypothesized that low CD10 antigen density patterns in males and in Blacks could be causally related to poor prognosis.

Unexpected cytokinetic effects induced by puromycin include a G2-arrest, a metaphase-mitotic-arrest, and apoptosis.

The potent effects of low doses of PM on the cell cycle have to date been obscured by the conventional usage of this drug at high concentrations (5-50 micrograms/ml) to inhibit protein synthesis. In this in vitro study undertaken in a variety of malignant and non-malignant human and murine cell types, we found that low doses of PM (0.1-0.5 microgram/ml) disrupted significant phase-to-phase cell cycle transitions, causing a G2-arrest, a metaphase-mitotic-arrest, and apoptosis. In HL-60 cells these observations were elicited by PM concentrations starting at 0.1 microgram/ml, and were more pronounced at slightly higher PM concentrations, including that (0.5 microgram/ml) which inhibited [14C]leucine incorporation by approximately 20% after one hour, and by approximately 50% after 24 h. A concentration of CHX (0.25 microgram/ml) which was equivalent to 0.5 microgram/ml of PM, both in terms of molarity (0.9 microM) and degree of inhibition of [14C]leucine incorporation, failed to induce similar changes to those induced by PM. This suggests that at these particular concentrations the PM-induced changes were likely to have been related to the different mechanisms of protein synthesis inhibition exerted by these two 'classical' translation inhibitors. PM but not CHX generates nascent peptidyl-PM complexes (PMPs), and we therefore propose that the subsequent intracellular effects exerted by the PMPs may account, in part, for the differential cytokinetic effects elicited by these drugs. The role of PM is currently being evaluated in vivo as a low-dose component of a multidrug chemotherapeutic regimen in which its cell cycle-specific effects could potentially be synergistic with other agents.

Puromycin is a potent and specific inhibitor of tyrosine kinase activity in HL-60 cells.

The search for specific tyrosine kinase inhibitors constitutes a novel approach to the development of anticancer agents. Puromycin (PM) inhibits protein synthesis by causing the premature release of truncated PM-peptide complexes (PMPs), the structure of which predicts an inhibitory effect on tyrosine kinase activity (21). The present study was undertaken to investigate the effects of a low dose of PM (0.9 microM) on tyrosine kinase activity in HL-60 leukaemic cells. Experiments were specifically controlled to exclude inhibitory effects consequent on protein synthesis blockade. Soluble enzyme extracts derived from PM-treated cells showed attenuated phosphorylation of two independent synthetic tyrosine kinase-specific substrates, the polymer Poly(Glu:Tyr; 4:1) and the peptide substrate Raytide. In contrast, Protein Kinase C-specific activity was unaffected by PM-exposure. It is possible that inhibition was due to metabolites of PM, perhaps PMPs, rather than to PM itself. Whether the inhibition of tyrosine kinase activity observed with the substrates used in this study is equally applicable to all tyrosine kinases, is not yet clear. Nevertheless, low-dose PM could provide a useful tool for dissecting out the role of tyrosine phosphorylation in the regulation and dysregulation of cell growth, and could conceivably prove beneficial in the treatment of tumours in which tyrosine kinase overactivity is linked to oncogenesis.